This invention relates to a method for a permanent fluorescent assay, and in particular, toward a fluorescent peroxidase-based assay utilizing a soluble p-hydroxyphenyl-containing substrate which forms an insoluble product through peroxidase activity.
Horseradish peroxidase (HRP) (EC 1.11.1.7) is a commonly used enzyme label for immunological detection systems. HRP decomposes two molecules of hydrogen peroxide into water and oxygen. HRP initiates this reaction when it donates a pair of electrons to hydrogen peroxide. The enzyme subsequently extracts electrons (oxidizes) from a suitable donor.
Donor substrates for HRP and other enzymes which form soluble fluorescent products have been described. Gross and Sizer, J. Biol. Chem., 234: 1611-1614 (1959) describe the mechanism of the oxidation of tyramine and tyrosine by peroxidase. They found that the reaction products of tyramine and tyrosine with peroxidase are dityramine and dityrosine, respectively. In their analysis, they demonstrated the presence of new soluble fluorescent substances.
Guilbault, et al., Analytical Chemistry, 40 (8): 1256-1263 (1980) describe new substrates for the fluorometric determination of oxidative enzymes. In an attempt to improve the sensitivity of the colorimetric determination of carbohydrates, Guilbault et al. evaluated a series of phenolic peroxidase substrates in conjunction with the oxidation of the carbohydrates by either galactose oxidase or glucose oxidase. The phenolic substrates were chosen because they formed fluorescent solution phase products upon reaction with peroxidase and H2O2. The best substrates were found to be p-hydroxyphenylacetic acid, homovanillic acid, tyramine and tyrosine. In characterizing the oxidized fluorescent products, Guilbault et al. reported an excitation range of 315-326 nm and emission range of 410-425 nm for the group.
Zaitsu and Ohkura, Analytical Biochemistry, 109: 109-113 (1980) reported new fluorogenic substrates for horseradish peroxidase. They evaluated twenty-five p-hydroxyphenyl or 3-methoxy-4-hydroxyphenyl compounds for their suitability as soluble fluorogenic substrates for HRP. They concluded that 3-(p-hydroxyphenyl) propionic acid was the best substrate. For the fluorescent assay employed, the range of excitation wavelengths was 312 to 320 nm and the range of emission wavelengths was 400-422 nm.
Non-enzymatic fluorescent detection methods have been used in assays but they often suffer from poor sensitivity, and when sensitive enough, are not stable and do not allow for the archiving of results (Raap, et al., Human Molecular Genetics, 4 (4): 529-534 (1995); Speel, et al., J. Histochem. Cytochem., 45 (10): 1439-1446 (1997); Chao et al., Cytometry, 23: 48-53 (1996). One method for enhancing sensitivity is the use of enzymatic amplification. For peroxidase based solid phase assays, this is made difficult due to the lack of soluble non-fluorescent substrates which react to form insoluble fluorescent products. Thus, there is a need for such substrates in enzymatically amplified assays since they would allow for enhanced sensitivity and for archival storage and later use of assay results.
This invention relates to a method for a permanent, highly fluorescent peroxidase-based assay comprising reacting peroxidase with a soluble p-hydroxyphenyl-containing substrate which forms an insoluble, fluorescent product.
In another embodiment, this invention is directed to a peroxidase-based assay for detecting the presence or absence of a desired substance in a sample which may contain the desired substance, wherein the method comprises reacting a peroxidase enzyme with a soluble p-hydroxyphenyl-containing substrate that forms an insoluble, fluorescent product through peroxidase activity.